Targeted gene modification using oligonucleotide-mediated gene repair

ABSTRACT

The invention provides to improved methods for the modification of genes in plant cells, and plants and seeds derived therefrom. More specifically, the invention relates to the increased efficiency of targeted gene mutation by combining gene repair oligonucleotides with approaches that enhance the availability of components of the target cell gene repair mechanisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of U.S. patent application Ser.No. 14/777,410, filed Sep. 15, 2015, now U.S. Pat. No. 11,434,494, whichis the U.S. national phase of International Application No.PCT/US2014/029621, filed Mar. 14, 2014, which designated the UnitedStates and claims priority to U.S. Provisional Application No.61/801,320, filed Mar. 15, 2013, each of which is hereby incorporated byreference in its entirety including all tables, figures, and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 5, 2022, isnamed CIBUS-026-CT1_SeqListing.txt and is 3 kilobytes in size.

FIELD OF THE INVENTION

This invention generally relates to novel methods to improve theefficiency of the targeting of modifications to specific locations ingenomic or other nucleotide sequences. Additionally, this inventionrelates to target DNA that has been modified, mutated or marked by theapproaches disclosed herein. The invention also relates to cells,tissue, and organisms which have been modified by the invention'smethods.

BACKGROUND OF THE INVENTION

DNA double-strand breaking (DSB) enhances homologous recombination inliving cells and has been exploited for targeted genome editing throughuse of engineered endonucleases. The key component of the engineerednucleases is the DNA recognition domain that is capable of directing thenuclease to the target site of genome for a genomic DNA double strandbreak. The cellular DSB repair due to nonhomologous end-joining (NHEJ)results in mutagenic deletions/insertions of a target gene. Alternately,the DSB can stimulate homologous recombination between the endogenoustarget locus and an exogenously introduced homologous DNA fragment withdesired genetic information, a process called gene targeting.

The most promising method involving gene or genome editing is thecustom-designed zinc finger nucleases (ZFN), a type of hybrid enzymeconsisting of DNA binding domains of zinc finger proteins and the FokInuclease domain (FN). The ZFN technology primarily involves the use ofhybrid proteins derived from the DNA binding domains of zinc finger (ZF)proteins and the nonspecific cleavage domain of the endonuclease FokI.The ZFs can be assembled as modules that are custom-designed torecognize selected DNA sequences following binding at the preselectedsite, a DSB is produced by the action of cleavage domain of FokI.

The FokI endonuclease was first isolated from the bacteriumFlavobacterium okeanokoites. This type US nuclease consists of twoseparate domains, the N-terminal DNA binding domain and C-terminal DNAcleavage domain. The DNA binding domain functions for recognition of anon-palindromic sequence 5′-GGATG-375′-CATCC-3′ while the catalyticdomain cleaves double-stranded DNA non-specifically at a fixed distanceof 9 and 13 nucleotides downstream of the recognition site. FokI existsas an inactive monomer in solution and becomes an active dimmerfollowing the binding to its target DNA and in the presence of somedivalent metals. As a functional complex, two molecules of FokI eachbinding to a double stranded DNA molecule dimerize through the DNAcatalytic domain for the effective cleavage of DNA double strands.

In a similar fashion, nucleases can also be made by using otherproteins/domains if they are capable of specific DNA recognition. TALeffectors belong to a large group of bacterial proteins that exist invarious strains of Xanthomonas and are translocated into host cells by atype III secretion system, so called type III effectors. Once in hostcells, some TAL effectors have been found to transcriptionally activatetheir corresponding host target genes either for strain virulence(ability to cause disease) or avirulence (capacity to trigger hostresistance responses) dependent on the host genetic context. Eacheffector contains the functional nuclear localization motifs and apotent transcription activation domain that are characteristic ofeukaryotic transcription activator. And each effector also contains acentral repetitive region consisting of varying numbers of repeat unitsof 34 amino acids, and the repeat region as DNA binding domaindetermines the biological specificity of each effector.

Zhang et al., Plant Physiol. 161: 20-27, 2013, which is herebyincorporated by reference in its entirety, discloses the use ofTALENs—transcriptional activator-like effector nucleases—which areengineered endonucleases based on the combination of a TAL effector-likeDNA binding domain with a catalytic domain of FokI. By engineering ofthe DNA binding domain, these TALENs reportedly can be easily designedto recognize specific DNA binding domains. Using tobacco protoplasts asa model system. TALEN activity was assessed using a single strandannealing polynucleotide reporter comprising a yellow fluorescentprotein coding sequence linked to a TALEN recognition site. Thisreporter system was delivered to protoplasts. and a cleavage-and-repairevent could be measured by expression of functional YFP.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to methods for introducing agene repair oligonucleobase (GRON)-mediated mutation into a targetdeoxyribonucleic acid (DNA) sequence in a plant cell. The methodscomprise, inter alia, culturing the plant cell under conditions thatincrease one or more cellular DNA repair processes prior to, and/orcoincident with, delivery of a GRON into the plant cell

In certain embodiments, the conditions that increase one or morecellular DNA repair processes comprise one or more of: introduction ofone or more sites into the GRON or into the plant cell DNA that aretargets for base excision repair, introduction of one or more sites intothe GRON or into the plant cell DNA that are targets for non-homologousend joining, introduction of one or more sites into the GRON or into theplant cell DNA that are targets for microhomology-mediated end joining,introduction of one or more sites into the GRON or into the plant cellDNA that are targets for homologous recombination, and introduction ofone or more sites into the GRON or into the plant cell DNA that aretargets for pushing repair.

In certain embodiments, the target deoxyribonucleic acid (DNA) sequenceis within the plant cell genome. The plant cell may be non-transgenic ortransgenic, and the target DNA sequence may be a transgene or anendogenous gene of the plant cell.

In certain embodiments, the conditions that increase one or morecellular DNA repair processes comprise introducing one or more compoundswhich induce single or double DNA strand breaks into the plant cellprior to or coincident with delivering the GRON into the plant cell.Exemplary compounds are described hereinafter.

The methods and compositions described herein are applicable to plantsgenerally. By way of example only, a plant species may be selected fromthe group consisting of canola, sunflower, corn, tobacco, sugar beet,cotton, maize, wheat, barley, rice, alfafa, barley, sorghum, tomato,mango, peach, apple, pear, strawberry, banana, melon, potato, carrot,lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, fieldpea, faba bean, lentils, turnip, rutabaga, brussel sprouts, lupin,cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus,triticale, alfalfa, rye, oats, turf and forage grasses, flax, oilseedrape, mustard, cucumber, morning glory, balsam, pepper, eggplant,marigold, lotus, cabbage, daisy, carnation, tulip, iris, and lily. Thesemay also apply in whole or in part to all other biological systemsincluding but not limited to bacteria, fungi and mammalian cells andeven their organelles (e.g., mitochondria and chloroplasts).

In certain embodiments, the methods further comprise regenerating aplant having a mutation introduced by the GRON from the plant cell, andmay comprise collecting seeds from the plant.

In related aspects, the present invention relates to plant cellscomprising a genomic modification introduced by a GRON according to themethods described herein, a plant comprising a genomic modificationintroduced by a GRON according to the methods described herein, or aseed comprising a genomic modification introduced by a GRON according tothe methods described herein.

Other embodiments of the invention will be apparent from the followingdetailed description, exemplary embodiments, and claims.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the effect of TALEN concentration on GRON targeting of aGFP to BFP mutation in an Arabidopsis protoplast model.

FIG. 2 depicts the design of a TALEN for use in targeting a BFP to GFPmutation as described in the Examples. Sequences depicted are:

(SEQ ID NO: 7; 5′-3′) CTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTGGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGG (SEQ ID NO: 5; 3′-5′)GAAGTACACCAGCCCCATCGCCGACTTCGTGACGTGCGGCACCCACTTCCACCAGTGCTCCCACCCGGTCCCGTGCCC (SEQ ID NO: 6; 3′-5′)KMHDPYRSFCQVGHTFTTVLTPWPVP.

FIG. 3 depicts the introduction of double strand breaks in a targetsequence mediate by TALEN plasmid pCLS14165 as measured by flowcytometry in a single strand annealing assay.

FIG. 4 depicts the percent GFP to BFP conversion mediated by TALENnickase (plasmid pCLS15771) alone, coding and non-coding GRON, and acombination of GRON plus TALEN.

FIG. 5 depicts the percent GFP to BFP conversion mediated by TALENnickase (plasmid pCLS15771) alone, coding and non-coding GRON, and acombination of GRON plus TALEN.

FIG. 6 depicts the percent GFP to BFP conversion mediated by TALENnickase (plasmid pCLS15771) alone, coding and non-coding GRON, and acombination of GRON plus TALEN.

FIG. 7 depicts the percent GFP to BFP conversion mediated by TALENnickase (plasmid pCLS15769) alone, coding and non-coding GRON, and acombination of GRON plus TALEN.

FIG. 8 depicts the percent GFP to BFP conversion mediated by TALENnickase (plasmid pCLS15769) alone, coding and non-coding GRON, and acombination of GRON plus TALEN.

FIG. 9 depicts the percent GFP to BFP conversion mediated by TALENnickase (plasmid pCLS15769) alone, coding and non-coding GRON, and acombination of GRON plus TALEN.

FIG. 10 depicts the percent GFP to BFP conversion mediated by TALENdouble strand breaker (plasmid pCLS14165) alone, coding and non-codingGRON, and a combination of GRON plus TALEN.

FIG. 11 depicts the percent GFP to BFP conversion mediated by TALENalone, coding and non-coding GRON, and a combination of GRON plus TALEN.

FIG. 12 depicts the percent GFP to BFP conversion mediated by TALENnickase (plasmid pCLS15769) alone, coding and non-coding GRON, and acombination of GRON plus TALEN.

FIG. 13 depicts the percent GFP to BFP conversion mediated by TALENnickase (plasmid pCLS15769) alone, coding and non-coding GRON, and acombination of GRON plus TALEN.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The invention is to be understood in accordance with the followingdefinitions.

An oligonucleobase is a polymer of nucleobases, which polymer canhybridize by Watson-Crick base pairing to a DNA having the complementarysequence.

Nucleobases comprise a base, which is a purine, pyrimidine, or aderivative or analog thereof. Nucleobases include peptide nucleobases,the subunits of peptide nucleic acids, and morpholine nucleobases aswell as nucleosides and nucleotides. Nucleosides are nucleobases thatcontain a pentosefuranosyl moiety, e.g., an optionally substitutedriboside or 2′-deoxyriboside. Nucleosides can be linked by one ofseveral linkage moieties, which may or may not contain a phosphorus.Nucleosides that are linked by unsubstituted phosphodiester linkages aretermed nucleotides.

An oligonucleobase chain has a single 5′ and 3′ terminus, which are theultimate nucleobases of the polymer. A particular oligonucleobase chaincan contain nucleobases of all types. An oligonucleobase compound is acompound comprising one or more oligonucleobase chains that arecomplementary and hybridized by Watson-Crick base pairing. Nucleobasesare either deoxyribo-type or ribo-type. Ribo-type nucleobases arepentosefuranosyl containing nucleobases wherein the 2′ carbon is amethylene substituted with a hydroxyl, alkyloxy or halogen.Deoxyribo-type nucleobases are nucleobases other than ribo-typenucleobases and include all nucleobases that do not contain apentosefuranosyl moiety.

An oligonucleobase strand generically includes both oligonucleobasechains and segments or regions of oligonucleobase chains. Anoligonucleobase strand has a 3′ end and a 5′ end. When a oligonucleobasestrand is coextensive with a chain, the 3′ and 5′ ends of the strand arealso 3′ and 5′ termini of the chain.

According to the present invention plant organs include, but are notlimited to, leaves, stems, roots, vegetative buds, floral buds,meristems, embryos, cotyledons, endosperm, sepals, petals, pistils,carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules,ovaries and fruits, or sections, slices or discs taken therefrom. Planttissues include, but are not limited to, callus tissues, ground tissues,vascular tissues, storage tissues, meristematic tissues, leaf tissues,shoot tissues, root tissues, gall tissues, plant tumor tissues, andreproductive tissues. Plant cells include, but are not limited to,isolated cells with cell walls, variously sized aggregates thereof, andprotoplasts.

Two polynucleotides or polypeptides are identical if the sequence ofnucleotides or amino acid residues, respectively, in the two sequencesis the same when aligned for maximum correspondence as described below.The terms “identical” or “percent identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Forpolypeptides where sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a ‘score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated according to, e.g.,the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17(1988) e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

The phrases “substantially identical,” and “percent identity” in thecontext of two nucleic acids or polypeptides, refer to sequences orsubsequences that have at least 50%, advantageously 60%, preferably 70%,more preferably 80%, and most preferably 90-95% nucleotide or amino acidresidue identity when aligned for maximum correspondence over acomparison window as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence, which hassubstantial sequence or subsequence complementarity when the testsequence has substantial identity to a reference sequence.

One of skill in the art will recognize that two polypeptides can also be“substantially identical” if the two polypeptides are immunologicallysimilar. Thus, overall protein structure may be similar while theprimary structure of the two polypeptides display significant variation.Therefore a method to measure whether two polypeptides are substantiallyidentical involves measuring the binding of monoclonal or polyclonalantibodies to each polypeptide. Two polypeptides are substantiallyidentical if the antibodies specific for a first polypeptide bind to asecond polypeptide with an affinity of at least one third of theaffinity for the first polypeptide. For sequence comparison, typicallyone sequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, 0.4dv. Appl. Math.2:482 (198 I), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Nat'I. Acad. Sci. USA 5 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), by software for alignments suchas VECTOR NTI Version #6 by InforMax, Inc. MD, USA, by the proceduresdescribed in ClustalW, Thompson, J. D., Higgins, D. G. and Gibson, T. J.(1994) CLUSTALW: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, position-specific gappenalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680or by visual inspection (see generally, Protocols in Molecular Biology,F. M. Ausubel et al., eds., Current Protocols, a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff& Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). Inaddition to calculating percent sequence identity, the BLAST algorithmalso performs a statistical analysis of the similarity between twosequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Strand Breakage

Inclusion of compounds which induce single or double strand breaks,either into the oligonucleotide or together with the oligonucleotide,generates a lesion which is repaired by non-homologous end joining(NHEJ), microhomology-mediated end joining (MMEJ), and homologousrecombination. By way of example, the bleomycin family of antibiotics,zinc finger nucleases, FokI (or any type US class of restriction enzyme)and other nucleases may be covalently coupled to the 3′ or 5′ end ofrepair oligonucleotides, in order to introduce double strand breaks inthe vicinity of the site targeted for conversion by the repairoligonucleotide. The bleomycin family of antibiotics are DNA cleavingglycopeptides include bleomycin, zeocin, phleomycin, tallysomycin,pepleomycin and others.

The invention's methods are not limited to the nature or type ofDNA-modifying reagent which is used. For example, such DNA-modifyingreagents release radicals which result in DNA strand breakage.Alternatively, the reagents alkylate DNA to form adducts which wouldblock replication and transcription. In another alternative, thereagents generate crosslinks or molecules that inhibit cellular enzymesleading to strand breaks. Examples of DNA-modifying reagents which havebeen linked to oligonucleotides to form TFOs include, but are notlimited to, indolocarbazoles, napthalene diimide (NDI), transplatin,bleomycin, analogues of cyclopropapyrroloindole, andphenanthodihydrodioxins. In particular, indolocarbazoles aretopoisomerase I inhibitors. Inhibition of these enzymes results instrand breaks and DNA protein adduct formation [Arimondo et al.,Bioorganic and Medicinal Chem. 8, 777, 2000]. NDI is a photooxidant thatcan oxidize guanines which could cause mutations at sites of guanineresidues [Nunez, et al., Biochemistry, 39, 6190, 2000]. Transplatin hasbeen shown to react with DNA in a triplex target when the TFO is linkedto the reagent. This reaction causes the formation of DNA adducts whichwould be mutagenic [Columbier, et al., Nucleic Acids Research, 24: 4519,1996]. Bleomycin is a DNA breaker, widely used as a radiation mimetic.It has been linked to oligonucleotides and shown to be active as abreaker in that format [Sergeyev, Nucleic Acids Research 23, 4400, 1995;Kane, et al., Biochemistry, 34, 16715, 1995]. Analogues ofcyclopropapyrroloindole have been linked to TFOs and shown to alkylateDNA in a triplex target sequence. The alkylated DNA would then containchemical adducts which would be mutagenic [Lukhtanov, et al., NucleicAcids Research, 25, 5077, 1997]. Phenanthodihydrodioxins are maskedquinones that release radical species upon photoactivation. They havebeen linked to TFOs and have been shown to introduce breaks into duplexDNA on photoactivation [Bendinskas et al., Bioconjugate Chem. 9, 555,1998].

One strategy for producing targeted gene disruption is through thegeneration of single strand or double strand DNA breaks caused bysite-specific endonucleases. Endonucleases are most often used fortargeted gene disruption in organisms that have traditionally beenrefractive to more conventional gene targeting methods, such as algae,plants, and large animal models, including humans. For example, thereare currently human clinical trials underway involving zinc fingernucleases for the treatment and prevention of HIV infection.Additionally, endonuclease engineering is currently being used inattempts to disrupt genes that produce undesirable phenotypes in crops.

The homing endonucleases, also known as meganucleases, are sequencespecific endonucleases that generate double strand breaks in genomic DNAwith a high degree of specificity due to their large (e.g., >14 bp)cleavage sites. While the specificity of the homing endonucleases fortheir target sites allows for precise targeting of the induced DNAbreaks, homing endonuclease cleavage sites are rare and the probabilityof finding a naturally occurring cleavage site in a targeted gene islow.

Engineered homing endonucleases are generated by modifying thespecificity of existing homing endonucleases. In one approach,variations are introduced in the amino acid sequence of naturallyoccurring homing endonucleases and then the resultant engineered homingendonucleases are screened to select functional proteins which cleave atargeted binding site. In another approach, chimeric homingendonucleases are engineered by combining the recognition sites of twodifferent homing endonucleases to create a new recognition site composedof a half-site of each homing endonuclease.

One class of artificial endonucleases is the zinc finger endonucleases.Zinc finger endonucleases combine a non-specific cleavage domain,typically that of FokI endonuclease, with zinc finger protein domainsthat are engineered to bind to specific DNA sequences. The modularstructure of the zinc finger endonucleases makes them a versatileplatform for delivering site-specific double-strand breaks to thegenome. One limitation of the zinc finger endonucleases is that lowspecificity for a target site or the presence of multiple target sitesin a genome can result in off-target cleavage events. As FokIendonuclease cleaves as a dimer, one strategy to prevent off-targetcleavage events has been to design zinc finger domains that bind atadjacent 9 base pair sites.

TALENs are targetable nucleases are used to induce single- anddouble-strand breaks into specific DNA sites, which are then repaired bymechanisms that can be exploited to create sequence alterations at thecleavage site.

The fundamental building block that is used to engineer the DNA-bindingregion of TALENs is a highly conserved repeat domain derived fromnaturally occurring TALEs encoded by Xanthomonas spp. proteobacteria.DNA binding by a TALEN is mediated by arrays of highly conserved 33-35amino acid repeats that are flanked by additional TALE-derived domainsat the amino-terminal and carboxy-terminal ends of the repeats.

These TALE repeats specifically bind to a single base of DNA, theidentity of which is determined by two hypervariable residues typicallyfound at positions 12 and 13 of the repeat, with the number of repeatsin an array corresponded to the length of the desired target nucleicacid, the identity of the repeat selected to match the target nucleicacid sequence. The target nucleic acid is preferably between 15 and 20base pairs in order to maximize selectivity of the target site. Cleavageof the target nucleic acid typically occurs within 50 base pairs ofTALEN binding. Computer programs for TALEN recognition site design havebeen described in the art. See, e.g., Cermak et al., Nucleic Acids Res.2011 July; 39(12): e82.

Once designed to match the desired target sequence, TALENS can beexpressed recombinantly and introduced into protoplasts as exogenousproteins, or expressed from a plasmid within the protoplast.

GRON Structure and Introduction into Plant Cells

The recombinagenic oligonucleobase can be introduced into a plant cellusing any method commonly used in the art, including but not limited to,microcarriers (biolistic delivery), microfibers (whiskers),electroporation, direct DNA uptake and microinjection. Illustrativeexamples of a recombinagenic oligonucleobase are described in FIGS. 4-13.

The invention can be practiced with recombinagenic oligonucleobaseshaving the conformations and chemistries described in the Kmiec I andKmiec II patents which are incorporated herein by reference. Kmiec Iteaches a method for introducing specific genetic alterations into atarget gene. The recombinagenic oligonucleobases in Kmiec I and/or KmiecII contain two complementary strands, one of which contains at least onesegment of RNA-type nucleotides (an “RNA segment”) that are base pairedto DNA-type nucleotides of the other strand.

Kmiec II discloses that purine and pyrimidine base-containingnon-nucleotides can be substituted for nucleotides. U.S. Pat. Nos.5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296;5,945,339; 6,004,804; and 6,010,907 and in International Patent No.PCT/US00/23457; and in International Patent Publication Nos. WO98/49350; WO 99/07865; WO 99/58723; WO 99/58702; WO 99/40789; U.S. Pat.No. 6,870,075; and US Published Patent Application 20030084473, whichare each hereby incorporated in their entirety, disclose additionalrecombinagenic molecules that can be used for the present invention. Theterm “recombinagenic oligonucleobase” is used herein to denote themolecules that can be used in the methods of the present invention andinclude mixed duplex oligonucleotides, non-nucleotide containingmolecules taught in Kmiec II, single stranded oligodeoxynucleotides andother recombinagenic molecules taught in the above noted patents andpatent publications.

In one embodiment, the recombinagenic oligonucleobase is a mixed duplexoligonucleotide in which the RNA-type nucleotides of the mixed duplexoligonucleotide are made RNase resistant by replacing the 2′-hydroxylwith a fluoro, chloro or bromo functionality or by placing a substituenton the 2′-O. Suitable substituents include the substituents taught bythe Kmiec II. Alternative substituents include the substituents taughtby U.S. Pat. No. 5,334,711 (Sproat) and the substituents taught bypatent publications EP 629 387 and EP 679 657 (collectively, the MartinApplications), which are incorporated herein by reference. As usedherein, a 2′-fluoro, chloro or bromo derivative of a ribonucleotide or aribonucleotide having a 2′-OH substituted with a substituent describedin the Martin Applications or Sproat is termed a “2′-SubstitutedRibonucleotide.” As used herein the term “RNA-type nucleotide” means a2′-hydroxyl or 2′-Substituted Nucleotide that is linked to othernucleotides of a mixed duplex oligonucleotide by an unsubstitutedphosphodiester linkage or any of the non-natural linkages taught byKmiec I or Kmiec II. As used herein the term “deoxyribo-type nucleotide”means a nucleotide having a 2′-H, which can be linked to othernucleotides of a MDON by an unsubstituted phosphodiester linkage or anyof the non-natural linkages taught by Kmiec I or Kmiec II.

In one embodiment of the present invention, the recombinagenicoligonucleobase is a mixed duplex oligonucleotide that is linked solelyby unsubstituted phosphodiester bonds. In alternative embodiments, thelinkage is by substituted phosphodiesters, phosphodiester derivativesand non-phosphorus-based linkages as taught by Kmiec II. In yet anotherembodiment, each RNA-type nucleotide in the mixed duplex oligonucleotideis a 2′-Substituted Nucleotide. Particularly preferred embodiments of2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy, 2′-propyloxy,2′-allyloxy, 2′-hydroxylethyloxy, 2′-methoxyethyloxy, 2′-fluoropropyloxyand 2′-trifluoropropyloxy substituted ribonucleotides. More preferredembodiments of 2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy,2′-methoxyethyloxy, and 2′-allyloxy substituted nucleotides. In anotherembodiment the mixed duplex oligonucleotide is linked by unsubstitutedphosphodiester bonds.

Although mixed duplex oligonucleotide having only a single type of2′-substituted RNA-type nucleotide are more conveniently synthesized,the methods of the invention can be practiced with mixed duplexoligonucleotides having two or more types of RNA-type nucleotides. Thefunction of an RNA segment may not be affected by an interruption causedby the introduction of a deoxynucleotide between two RNA-typetrinucleotides, accordingly, the term RNA segment encompasses such an“interrupted RNA segment.” An uninterrupted RNA segment is termed acontiguous RNA segment. In an alternative embodiment an RNA segment cancontain alternating RNase-resistant and unsubstituted 2′-OH nucleotides.The mixed duplex oligonucleotides preferably have fewer than 100nucleotides and more preferably fewer than 85 nucleotides, but more than50 nucleotides. The first and second strands are Watson-Crick basepaired. In one embodiment the strands of the mixed duplexoligonucleotide are covalently bonded by a linker, such as a singlestranded hexa, penta or tetranucleotide so that the first and secondstrands are segments of a single oligonucleotide chain having a single3′ and a single 5′ end. The 3′ and 5′ ends can be protected by theaddition of a “hairpin cap” whereby the 3′ and 5′ terminal nucleotidesare Watson-Crick paired to adjacent nucleotides. A second hairpin capcan, additionally, be placed at the junction between the first andsecond strands distant from the 3′ and 5′ ends, so that the Watson-Crickpairing between the first and second strands is stabilized.

The first and second strands contain two regions that are homologouswith two fragments of the target ACCase gene, i.e., have the samesequence as the target gene. A homologous region contains thenucleotides of an RNA segment and may contain one or more DNA-typenucleotides of connecting DNA segment and may also contain DNA-typenucleotides that are not within the intervening DNA segment. The tworegions of homology are separated by, and each is adjacent to, a regionhaving a sequence that differs from the sequence of the target gene,termed a “heterologous region.” The heterologous region can contain one,two or three mismatched nucleotides. The mismatched nucleotides can becontiguous or alternatively can be separated by one or two nucleotidesthat are homologous with the target gene. Alternatively, theheterologous region can also contain an insertion or one, two, three orof five or fewer nucleotides. Alternatively, the sequence of the mixedduplex oligonucleotide may differ from the sequence of the target geneonly by the deletion of one, two, three, or five or fewer nucleotidesfrom the mixed duplex oligonucleotide. The length and position of theheterologous region is, in this case, deemed to be the length of thedeletion, even though no nucleotides of the mixed duplex oligonucleotideare within the heterologous region. The distance between the fragmentsof the target gene that are complementary to the two homologous regionsis identically the length of the heterologous region when a substitutionor substitutions is intended. When the heterologous region contains aninsertion, the homologous regions are thereby separated in the mixedduplex oligonucleotide farther than their complementary homologousfragments are in the gene, and the converse is applicable when theheterologous region encodes a deletion.

The RNA segments of the mixed duplex oligonucleotides are each a part ofa homologous region, i.e., a region that is identical in sequence to afragment of the target gene, which segments together preferably containat least 13 RNA-type nucleotides and preferably from 16 to 25 RNA-typenucleotides or yet more preferably 18-22 RNA-type nucleotides or mostpreferably 20 nucleotides. In one embodiment, RNA segments of thehomology regions are separated by and adjacent to, i.e., “connected by”an intervening DNA segment. In one embodiment, each nucleotide of theheterologous region is a nucleotide of the intervening DNA segment. Anintervening DNA segment that contains the heterologous region of a mixedduplex oligonucleotide is termed a “mutator segment.”

The change to be introduced into the target gene is encoded by theheterologous region. The change to be introduced into the Agene may be achange in one or more bases of the gene sequence that changes the nativeamino acid in that position to the desired amino acid.

In another embodiment of the present invention, the recombinagenicoligonucleobase is a single stranded oligodeoxynucleotide mutationalvector or SSOMV, which is disclosed in International Patent ApplicationPCT/US00/23457, which is incorporated herein by reference in itsentirety. The sequence of the SSOMV is based on the same principles asthe mutational vectors described in U.S. Pat. Nos. 5,756,325; 5,871,984;5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and6,010,907 and in International Publication Nos. WO 98/49350; WO99/07865; WO 99/58723; WO 99/58702; WO 99/40789; U.S. Pat. No.6,870,075; and US Published Patent Application 20030084473. The sequenceof the SSOMV contains two regions that are homologous with the targetsequence separated by a region that contains the desired geneticalteration termed the mutator region. The mutator region can have asequence that is the same length as the sequence that separates thehomologous regions in the target sequence, but having a differentsequence. Such a mutator region will cause a substitution.

The nucleotides of the SSOMV are deoxyribonucleotides that are linked byunmodified phosphodiester bonds except that the 3′ terminal and/or 5′terminal internucleotide linkage or alternatively the two 3′ terminaland/or 5′ terminal internucleotide linkages can be a phosphorothioate orphosphoamidate. As used herein an internucleotide linkage is the linkagebetween nucleotides of the SSOMV and does not include the linkagebetween the 3′ end nucleotide or 5′ end nucleotide and a blockingsubstituent, see supra. In a specific embodiment the length of the SSOMVis between 21 and 55 deoxynucleotides and the lengths of the homologyregions are, accordingly, a total length of at least 20 deoxynucleotidesand at least two homology regions should each have lengths of at least 8deoxynucleotides.

The SSOMV can be designed to be complementary to either the coding orthe non-coding strand of the target gene. When the desired mutation is asubstitution of a single base, it is preferred that both the mutatornucleotides be a pyrimidine. To the extent that is consistent withachieving the desired functional result it is preferred that both themutator nucleotide and the targeted nucleotide in the complementarystrand be pyrimidines. Particularly preferred are SSOMV that encodetransversion mutations, i.e., a C or T mutator nucleotide is mismatched,respectively, with a C or T nucleotide in the complementary strand.

In addition to the oligodeoxynucleotide the SSOMV can contain a 5′blocking substituent that is attached to the 5′ terminal carbons througha linker. The chemistry of the linker is not critical other than itslength, which should preferably be at least 6 atoms long and that thelinker should be flexible. A variety of non-toxic substituents such asbiotin, cholesterol or other steroids or a non-intercalating cationicfluorescent dye can be used. Particularly preferred as reagents to makeSSOMV are the reagents sold as Cy3™ and Cy5™ by Glen Research, SterlingVa. (now GE Healthcare), which are blocked phosphoroamidites that uponincorporation into an oligonucleotide yield 3,3,3′,3′-tetramethylN,N′-isopropyl substituted indomonocarbocyanine and indodicarbocyaninedyes, respectively. Cy3 is the most preferred. When the indocarbocyanineis N-oxyalkyl substituted it can be conveniently linked to the 5′terminal of the oligodeoxynucleotide through as a phosphodiester with a5′ terminal phosphate. The chemistry of the dye linker between the dyeand the oligodeoxynucleotide is not critical and is chosen for syntheticconvenience. When the commercially available Cy3 phosphoramidite is usedas directed the resulting 5′ modification consists of a blockingsubstituent and linker together which are a N-hydroxypropyl,N′-phosphatidylpropyl 3,3,3′,3′-tetramethyl indomonocarbocyanine.

In a preferred embodiment the indocarbocyanine dye is tetra substitutedat the 3 and 3′ positions of the indole rings. Without limitation as totheory these substitutions prevent the dye from being an intercalatingdye. The identity of the substituents at these positions are notcritical. The SSOMV can in addition have a 3′ blocking substituent.Again the chemistry of the 3′ blocking substituent is not critical.

In another preferred embodiment the recombinageneic oligonucleotide is asingle-stranded oligodeoxynucleotide having a 3′ end nucleotide, a 5′end nucleotide, having at least 25 deoxynucleotides and not more than 65deoxynucleotides, and having a sequence comprising at least two regionseach of at least 8 deoxynucleotides that are each, respectively,identical to at least two regions of the targeted chromosomal gene,which regions together are at least 24 nucleotides in length, and whichregions are separated by at least one nucleotide in the sequence of thetargeted chromosomal gene or in the sequence of the oligodeoxynucleotideor both such that the sequence of the oligodeoxynucleotide is notidentical to the sequence of the targeted chromosomal gene. See U.S.Pat. No. 6,271,360 which is incorporated herein by reference.

Microcarriers and Microfibers

The use of metallic microcarriers (microspheres) for introducing largefragments of DNA into plant cells having cellulose cell walls byprojectile penetration is well known to those skilled in the relevantart (henceforth biolistic delivery). U.S. Pat. Nos. 4,945,050; 5,100,792and 5,204,253 describe general techniques for selecting microcarriersand devices for projecting them. U.S. Pat. Nos. 5,484,956 and 5,489,520describe the preparation of fertile transgenic corn usingmicroprojectile bombardment of corn callus tissue. The biolistictechniques are also used in transforming immature corn embryos.

Specific conditions for using microcarriers in the methods of thepresent invention are described in International Publication WO99/07865. In an illustrative technique, ice cold microcarriers (60mg/ml), mixed duplex oligonucleotide (60 mg/ml) 2.5 M CaCl₂) and 0.1 Mspermidine are added in that order; the mixture is gently agitated,e.g., by vortexing, for 10 minutes and let stand at room temperature for10 minutes, whereupon the microcarriers are diluted in 5 volumes ofethanol, centrifuged and resuspended in 100% ethanol. Good results canbe obtained with a concentration in the adhering solution of 8-10 μg/μlmicrocarriers, 14-17 μg/ml mixed duplex oligonucleotide, 1.1-1.4 M CaC₂and 18-22 mM spermidine. Optimal results were observed under theconditions of 8 μg/μl microcarriers, 16.5 μg/ml mixed duplexoligonucleotide, 1.3 M CaCl and 21 mM spermidine.

Recombinagenic oligonucleobases can also be introduced into plant cellsfor the practice of the present invention using microfibers to penetratethe cell wall and cell membrane. U.S. Pat. No. 5,302,523 to Coffee etal. describes the use of 30.times.0.5 μm and 10.times.0.3 μm siliconcarbide fibers to facilitate transformation of suspension maize culturesof Black Mexican Sweet. Any mechanical technique that can be used tointroduce DNA for transformation of a plant cell using microfibers canbe used to deliver recombinagenic oligonucleobases for use in making thepresent ACCase mutants. An illustrative technique for microfiberdelivery of a recombinagenic oligonucleobase is as follows: Sterilemicrofibers (2 μg) are suspended in 150 μl of plant culture mediumcontaining about 10 μg of a mixed duplex oligonucleotide. A suspensionculture is allowed to settle and equal volumes of packed cells and thesterile fiber/nucleotide suspension are vortexed for 10 minutes andplated. Selective media are applied immediately or with a delay of up toabout 120 hours as is appropriate for the particular trait.

Electroporation

In an alternative embodiment, the recombinagenic oligonucleobases can bedelivered to the plant cell by electroporation of a protoplast derivedfrom a plant part according to techniques that are well-known to one ofordinary skill in the art. See, e.g., Gallois et al., 1996, in Methodsin Molecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp et al.,1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa,N.J.

Recombinagenic oligonucleobases can also be introduced into microsporesby electroporation. Upon release of the tetrad, the microspore isuninucleate and thin-walled. It begins to enlarge and develops agermpore before the exine forms. A microspore at this stage ispotentially more amenable to transformation with exogenous DNA thanother plant cells. In addition, microspore development can be altered invitro to produce either haploid embryos or embryogenic callus that canbe regenerated into plants (Coumans et al., Plant Cell Rep. 7:618-621,1989; Datta et al., Plant Sci. 67:83-88, 1990; Maheshwari et al., Am. JBot. 69:865-879, 1982; Schaeffer, Adv. In Cell Culture 7:161-182, 1989;Swanson et al., Plant Cell Rep. 6:94-97, 1987). Thus, transformedmicrospores can be regenerated directly into haploid plants or dihaploidfertile plants upon chromosome doubling by standard methods. See alsoco-pending application U.S. Ser. No. 09/680,858 entitled Compositionsand Methods for Plant Genetic Modification which is incorporated hereinby reference.

Microspore electroporation methods are described in Jardinaud et al.,Plant Sci. 93:177-184, 1993, and Fennell and Hauptman, Plant CellReports 11:567-570, 1992. Methods for electroporation of MDON into plantprotoplasts can also be adapted for use in microspore electroporation.

Whiskers and Microinjection

In yet another alternative embodiment, the recombinagenicoligonucleobase can be delivered to the plant cell by whiskers ormicroinjection of the plant cell. The so called whiskers technique isperformed essentially as described in Frame et al., 1994, Plant J.6:941-948. The recombinagenic oligonucleobase is added to the whiskersand used to transform the plant cells. The recombinagenicoligonucleobase may be co-incubated with plasmids comprising sequencesencoding proteins capable of forming recombinase complexes in plantcells such that recombination is catalyzed between the oligonucleotideand the target sequence.

Selection of Plants

In various embodiments, plants as disclosed herein can be of any speciesof dicotyledonous, monocotyledonous or gymnospermous plant, includingany woody plant species that grows as a tree or shrub, any herbaceousspecies, or any species that produces edible fruits, seeds orvegetables, or any species that produces colorful or aromatic flowers.For example, the plant maybe selected from a species of plant from thegroup consisting of canola, sunflower, corn, tobacco, sugar beet,cotton, maize, wheat, barley, rice, alfafa, barley, sorghum, tomato,mango, peach, apple, pear, strawberry, banana, melon, potato, carrot,lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, fieldpea, faba bean, lentils, turnip, rutabaga, brussel sprouts, lupin,cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus,triticale, alfalfa, rye, oats, turf and forage grasses, flax, oilseedrape, mustard, cucumber, morning glory, balsam, pepper, eggplant,marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nutproducing plants insofar as they are not already specifically mentioned.

Plants and plant cells can be tested for resistance or tolerance to anherbicide using commonly known methods in the art, e.g., by growing theplant or plant cell in the presence of an herbicide and measuring therate of growth as compared to the growth rate in the absence of theherbicide.

As used herein, substantially normal growth of a plant, plant organ,plant tissue or plant cell is defined as a growth rate or rate of celldivision of the plant, plant organ, plant tissue, or plant cell that isat least 35%, at least 50%, at least 60%, or at least 75% of the growthrate or rate of cell division in a corresponding plant, plant organ,plant tissue or plant cell expressing the wild-type AHAS protein.

As used herein, substantially normal development of a plant, plantorgan, plant tissue or plant cell is defined as the occurrence of one ormore development events in the plant, plant organ, plant tissue or plantcell that are substantially the same as those occurring in acorresponding plant, plant organ, plant tissue or plant cell expressingthe wild-type protein.

In certain embodiments plant organs provided herein include, but are notlimited to, leaves, stems, roots, vegetative buds, floral buds,meristems, embryos, cotyledons, endosperm, sepals, petals, pistils,carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules,ovaries and fruits, or sections, slices or discs taken therefrom. Planttissues include, but are not limited to, callus tissues, ground tissues,vascular tissues, storage tissues, meristematic tissues, leaf tissues,shoot tissues, root tissues, gall tissues, plant tumor tissues, andreproductive tissues. Plant cells include, but are not limited to,isolated cells with cell walls, variously sized aggregates thereof, andprotoplasts.

Plants are substantially “tolerant” to a relevant herbicide when theyare subjected to it and provide a dose/response curve which is shiftedto the right when compared with that provided by similarly subjectednon-tolerant like plant. Such dose/response curves have “dose” plottedon the X-axis and “percentage kill”, “herbicidal effect”, etc., plottedon the y-axis. Tolerant plants will require more herbicide thannon-tolerant like plants in order to produce a given herbicidal effect.Plants that are substantially “resistant” to the herbicide exhibit few,if any, necrotic, lytic, chlorotic or other lesions, when subjected toherbicide at concentrations and rates which are typically employed bythe agrochemical community to kill weeds in the field. Plants which areresistant to an herbicide are also tolerant of the herbicide.

Generation of Plants

Tissue culture of various tissues of plant species and regeneration ofplants therefrom is known. For example, the propagation of a canolacultivar by tissue culture is described in any of the following but notlimited to any of the following: Chuong et al., “A Simple Culture Methodfor Brassica hypocotyls Protoplasts,” Plant Cell Reports 4:4-6, 1985;Barsby, T. L., et al., “A Rapid and Efficient Alternative Procedure forthe Regeneration of Plants from Hypocotyl Protoplasts of Brassicanapus,” Plant Cell Reports (Spring, 1996); Kartha, K., et al., “In vitroPlant Formation from Stem Explants of Rape,” Physiol. Plant, 31:217-220,1974; Narasimhulu, S., et al., “Species Specific Shoot RegenerationResponse of Cotyledonary Explants of Brassicas,” Plant Cell Reports(Spring 1988); Swanson, E., “Microspore Culture in Brassica,” Methods inMolecular Biology, Vol. 6, Chapter 17, p. 159, 1990.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Komatsuda, T. et al., “Genotype XSucrose Interactions for Somatic Embryogenesis in Soybeans,” Crop Sci.31:333-337, 1991; Stephens, P. A., et al., “Agronomic Evaluation ofTissue-Culture-Derived Soybean Plants,” Theor. Appl. Genet. 82:633-635,1991; Komatsuda, T. et al., “Maturation and Germination of SomaticEmbryos as Affected by Sucrose and Plant Growth Regulators in SoybeansGlycine gracilis Skvortz and Glycine max (L.) Merr.” Plant Cell, Tissueand Organ Culture, 28:103-113, 1992; Dhir, S. et al., “Regeneration ofFertile Plants from Protoplasts of Soybean (Glycine max L. Merr.);Genotypic Differences in Culture Response,” Plant Cell Reports11:285-289, 1992; Pandey, P. et al., “Plant Regeneration from Leaf andHypocotyl Explants of Glycine wightii (W. and A.) VERDC. var.longicauda,” Japan J. Breed. 42:1-5, 1992; and Shetty, K., et al.,“Stimulation of In Vitro Shoot Organogenesis in Glycine max (Merrill.)by Allantoin and Amides,” Plant Science 81:245-251, 1992. Thedisclosures of U.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collinset al., and U.S. Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch etal., are hereby incorporated herein in their entirety by reference.

The following example and FIGS. 4-13 illustrate the practice of thepresent invention but should not be construed as limiting its scope.

Example 1: Dramatically improved conversion of a blue fluorescentprotein (BFP) gene in transgenic Arabidopsis thaliana cells to a greenfluorescing protein (GFP) by introducing a targeted single nucleotidemutation through Gene Repair Oligonucleotides (GRONs) in combinationwith a Transcription Activator-Like Effector Nuclease (TALEN) pair thatcleaves near the targeted base change.

An Arabidopsis line with multiple copies of a blue fluorescent proteingene was created by methods known to those skilled in the art (see,e.g., Clough and Brent, 1998). Root-derived meristematic tissue cultureswere established with this line, which was used for protoplast isolationand culture (see, e.g., Mathur et al., 1995). GRON delivery intoprotoplasts was achieved through polyethylene glycol (PEG) mediated GRONuptake into protoplasts. A method using a 96-well format, similar tothat described by similar to that described by Fujiwara and Kato (2007)was used. In the following the protocol is briefly described. Thevolumes given are those applied to individual wells of a 96-well dish.

-   -   1. Mix 6.25 μl of GRON/TALEN mix (80 μM BFP4 Coding/41mer GRON)        with 25 μl of Arabidopsis BFP transgenic root meristematic        tissue-derived protoplasts at 5×10⁶ cells/ml in each well of a        96 well plate.    -   2. 31.25 μl of a 40% PEG solution was added and the protoplasts        were mixed.    -   3. Treated cells were incubated on ice for 30 min.    -   4. To each well 200 μl of W5 solution was added and the cells        mixed.    -   5. The plates were allowed to incubate on ice for 30 min        allowing the protoplasts to settle to the bottom of each well.    -   6. 200 μl of the medium above the settled protoplasts was        removed.    -   7. 85 μl of culture medium (MSAP, see Mathur et al., 1995) was        added.    -   8. The plates were incubated at room temperate in the dark for        48 hours. The final concentration of GRON after adding culture        medium is 8 μM.

Using this protocol, TALEN plasmids at different concentrations wereintroduced together with GRON. Forty eight hours after GRON deliverysamples were analyzed by flow cytometry in order to detect protoplastswhose green and yellow fluorescence is different from that of controlprotoplasts. The green fluorescence is caused by the introduction of atargeted mutation in the BFP gene, resulting in the synthesis of GFP.The results are shown in FIG. 1 .

FIG. 1 . Root meristematic tissue-derived protoplasts were treated withTALEN plasmids at various concentrations together with GRON targeting amutation in the BFP gene, causing a conversion into a GFP gene. GFPexpression was measured by flow cytometry 48 h after GRON/TALEN delivery

For FIGS. 4-13 , the following Legend applies:

GRONs

BFP->GFP targeting designs.

BFP->GFP H66Y CAC->TAC. BFP4/C/41/5′Cy3/3′idC (SEQ ID NO: 1):VCCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCHBFP4/NC/41/5′Cy3/3′idC (SEQ ID NO: 2):VGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGH

BFP non-targeting control designs. PGP-22.DNA

BFP H66-CAC BFPO/C/41/5′3PS/3′3PS (SEQ ID NO: 3):VCCCTCGTGACCACCTTCACCCACGGCGTGCAGTGCTTCAGCHBFP0/NC/41/5′3PS/3′3PS (SEQ ID NO: 4):VGCTGAAGCACTGCACGCCGTGGGTGAAGGTGGTCACGAGGGH

pCLS14165 has both TAL arms (designed as per Zhang et al., 2013) on asingle plasmid with each arm binding to the underlined sequence andlinked to a FokI monomer. This combination produces a double strandbreak (DSB) as shown in FIG. 3 in the single strand annealing assay(SSA) performed in the same was as the ones in Zhang et al. (2013)

pCLS15771 is a nickase with a mutation (D450A) in the FokI domain forthe left arm. The right arm in this construct is as per pCLS14165.

pCLS15769 is a nickase with a mutation (D450A) in the FokI domain forthe right arm. The left arm in this construct is as per pCLS14165.

The GRONs and TALENs were tested as shown in FIGS. 4-13 . The controltreatments consisting of non-targeting GRONs (BFP0/C or BFP0/NC) andTALENs alone as well as mock treatments with the 40% PEG solutionlacking GRONs or TALEN plasmid had no significant conversion activity.

In this system the BFP4/NC GRON design alone is better than the BFP4/CGRON design alone. Combining these with the DSB TALEN (pCLS14165)improves both with the best activity and fold improvement (in manycases>2 orders of magnitude) with the BFP4/C GRON. Significantimprovement are also observed in combining GRONs with nickase TALENpairs and are expected to be most beneficial by minimizing collateraldamage when mutations are targeted in several genes/loci/allelessimultaneously.

REFERENCES

-   Clough, S. J., and Bent, A. F. (1998). Floral dip: A simplified    method for Agrobacterium-mediated transformation of Arabidopsis    thaliana. Plant J. 16, 735-743.-   Mathur, J., Szabados, L, and Koncz, C. (1995) A simple method for    isolation, liquid culture, transformation and regeneration of    Arabidopsis thaliana protoplasts. Plant Cell Rep. 14, 221-226-   Fujikawa Y, Kato N (2007) Split luciferase complementation assay to    study protein-protein interactions in Arabidopsis protoplasts. Plant    J 52: 185-195-   Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ,    Voytas DF. (2013) Transcription activator-like effector nucleases    enable efficient plant genome engineering. Plant Physiol.    161(1):20-7.

1. A method for introducing a gene repair oligonucleobase(GRON)-mediated mutation into a target deoxyribonucleic acid (DNA)sequence in a cell, comprising: culturing the cell under conditions thatincrease one or more cellular DNA repair processes prior to, and/orcoincident with, delivery of a GRON into the plant cell.